Covalent attachment of antibodies and antigens to solid phases using extended length heterobifunctional coupling agents

ABSTRACT

Novel methods for covalent attachment of antibodies, antigens, or other molecules to solid phases using extended length heterobifunctional crosslinking reagents are disclosed. The resulting derivatized solid phases can be used in diagnostic assays.

This is a division of application Ser. No. 254,288, filed Oct. 11, 1988,now U.S. Pat. No. 5,002,883, which in turn is a continuation in part ofU.S. patent application Ser. No. 114,930 filed Oct. 30, 1987, nowabandoned, entitled Heterobifunctional Coupling Agent which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a method for covalent attachment ofantibodies or other molecules to a solid support using extended lengthheterobifunctional reagents.

In diagnostic assays, the reaction between a specific binding member andits complement is often employed to detect whether (and in some assays,how much) specific binding member or complement is present in a sample.In one type of diagnostic assay, a specific binding member (e.g. anantibody) is detected in a sample by introducing its complement (e.g. anantigen) into the sample and determining if any reaction occurs betweenthe two reagents. Alternatively, the complement itself can be detectedin a sample by introducing the specific binding member into the sampleand determining whether any reaction occurs. Because it is oftendifficult to detect whether any reaction has occurred, a second specificbinding member may be added to the sample. The second specific bindingmember can react either with the first specific binding member or itscomplement, and the second member bears a detectable label. Of course,it is impossible to determine beforehand how much labelled secondspecific binding member must be added because it is unknown how much, ifany, of the substance to be detected is in the sample. Thus, thelabelled specific binding member is added in excess of the maximumconcentration of the substance typically found in such samples. However,the labelled specific binding member which does not bind with thesubstance must be separated from the sample so that only the boundlabelled member is detected, indicating that the substance is indeed inthe sample.

A common approach to separate bound from unbound labelled member is toemploy solid phase separation. A typical example of such separationinvolves linking the first specific binding member (in the case ofassays for complement to the first member) or complement (in the case ofassays for specific binding member) to a solid phase (such asmicroparticles) which can be separated from the sample, for example, byfiltration or gravity sedimentation. The label associated either withthe solid phase or still in the sample is proportional to the amount ofsubstance to be detected in the original sample.

Other variations to this general solid phase separation scheme have beendeveloped, but most such schemes involve the binding of the substance tobe analyzed to a specific binding member linked to a solid phase. Thisbinding is crucial to assay performance. However, the linkage betweenthe solid phase and the specific binding member can subsequently affectbinding of the substance to be analyzed. An example will illustrate thepoint. Antibodies have extremely specific structural, spacial, and polarconfigurations which endow them with the ability to recognize and bindto one type of analyte, and virtually none other. When antibodies areemployed in assays for the detection of antigens, antibodies can belinked to solid phases. However, the proximity of the solid phase to theantibody can block sites on the antibody where antigen binds.Alternatively, the linkage (usually covalent) between the antibody andsolid phase can alter the structure (conformation) of the antibody sothat the linkage may deleteriously affect binding of the antibody to theanalyte. The same situation holds for conjugation of analytes,particularly proteins, to solid phases. The analyte conformation canchange upon conjugation so the free antibody in the sample can no longerrecognize it.

Covalent attachment of proteins to solid phases using heterobifunctionalreagents has been accomplished with mixed results in the past. In somecases the proteins were directly conjugated to the solid phase.Generally, the connecting tether has been quite short in comparison withthe size of the bound protein. This is disadvantageous in that the boundprotein can still be hindered in performing its biological function dueto steric crowding, inaccessability of binding sites, etc. This has beena problem which has limited the bioactivity and stability of derivatizedsolid phases in the past.

SUMMARY OF THE INVENTION

This invention involves conjugates of solid phases with novel linkinggroups which can be used to link solid phases to substances such asproteins, antibodies, antigens and the like. These conjugates arehydrophyllic, so they tend to be quite stable in aqueous solutions, andthey preserve the conformation of such substances when such substancesare linked to solid phases. At the same time, these linking groups areof such lengths that the solid phases tend not to interfere with bindingsites on such substances.

This invention involves derivatized solid phase materials of theformula: ##STR1## wherein B is an amine bearing solid phase material; Xis a substituted or unsubstituted amino acid having from three to tencarbon atoms in a straight chain; n is from one to ten; and R is analkyl, cycloalkyl, an alkyl cycloalkyl or an aromatic carbocyclic ring.

The current invention also involves protein solid phase conjugates ofthe formula: ##STR2## wherein Q is a SH or a thiol bearing peptide,polypeptide or protein; and wherein B, X, n, and R are as definedpreviously.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above, the present invention involves conjugates of "aminebearing solid phases." Such solid phases include those polymers,glasses, and natural products which bear amine (either primary,secondary, or tertiary) groups which can be reacted with a maleimidemoiety to form a stable covalent bond. A wide variety of solid phasesare possible, consistent with this definition: commercially availablepolystyrene aminated particles, amino silica gels, partially hydrolyzednylon, partially reduced polyacrylamides, partially reducedcyanoacrylates and copolymers containing such polymers. Solid phasescontaining nitrile groups which can be reduced to yield amine groups toproduce "amine-bearing solid phases" consistent with this invention.

While the Examples which follow generally deal with microparticle solidphases, other solid phase configurations are possible: beads, sheets,spheres, filters, and the like. However, one solid phase configurationof particular interest is fibers. Many of the polymers mentionedpreviously are available in fiberous form. These fibers can be chopped(discontinuous) or continuous, but the latter are preferred. Continuousfibers can be derivatized with the linking groups of this invention, andproteins such as antibodies can be conjugated to the linking groups. Theprotein-bearing fiber can then be sewn or woven into a solid supportsuch as cloth, a mat, or a woven or non woven filter media. The fibercan be sewn or woven into distinctive patterns. For example, the fiberscan be arranged in a plus (+) sign with the vertical bar having positivecontrols and the horizontal bar having negative controls, as disclosedin copending U.S. application Ser. No. 831,013, filed Feb. 18, 1986, nowabandoned, which is incorporated herein by reference. Thus, when anassay is performed using the sewn fibers, a plus sign will appear if thesample passed through the media has analyte in it, and a minus (-)(horizontal bar only) will appear if no analyte is in the sample.

Another approach is to attach the linking groups of this invention tothe fiber, sew the fiber into an inert backing material (i.e. a materialwhich lacks maleimide groups), and react the protein to the exposedmaleimide moieties on the linking groups.

The term "alkyl-cycloalkyl" as used herein includes alkyl groups linkedto cycloalkyl ring structures where the alkyl group links the cycloalkylto the maleimide or the carbonyl groups in the chemical structures shownabove. The term "alkyl" includes straight or branched alkyl groups,preferably lower alkyl groups having from one to six carbon atoms.

The substituent "Q" is a --SH or thiol bearing peptide, polypeptide orprotein. For convenient reaction with the linking groups of thisinvention, the peptide, polypeptide or protein to be conjugated to thelinking groups bear reactive thiol (mercapto) or sulfhydryl (--SH)groups. It is recognized that mercapto groups contain sulfhydryl groups,but this invention contemplates that peptides, polypeptides or proteinslacking sulfhydryl groups may be artificially derivatized withsulfhydryl groups which are not mercapto groups, mercapto groups beingorganic compounds bearing sulfhydryl groups. The sulfhydryl group on thepeptide, polypeptide or protein reacts with maleimide moiety on thelinking groups of this invention to form a covalent bond between thelinking groups and the peptide, polypeptide or protein.

The Examples which follow illustrate this invention. Examples 1-6describe the synthesis of various linker groups of this invention.Examples 7-18 describe the uses of the linker groups to conjugate solidphases to proteins. These examples are not intended to limit theinvention.

EXAMPLE 1 ##STR3##

Trans-4-(aminomethyl)-cyclohexanecarboxylic acid was purchased fromAldrich Chemical Co. and converted to N-(4-carboxycyclohexylmethyl)maleimide N-Hydroxysuccinimide active ester by the method of Yoshitakeet al. (J. Biochem., 101:395-399 (1979)). This material (100 mg) is thendissolved in dry dimethylformamide (DMF) (1.0 ml), 6-aminocaproic acid(39.2 mg; 1.0 eg) is added, and the resulting mixture is stirredovernight at room temperature under nitrogen atmosphere. The followingmorning dicyclohexylcarbodiimide (DCCI) (67.8 mg; 1.1 eq) is added, andthe reaction mixture is stirred for an additional six hours.Precipitated dicyclohexylurea (DCU) is removed by filtration, and theresulting DMF solution is evaporated under reduced pressure to give atacky solid, which is purified by flash chromatography upon silica gel(5% methanol/chloroform) to give compound 1 (71 mg) as a white solid in53% overall yield. (R=cyclohexylmethyl; n=1; X=6-aminocaproyl).

EXAMPLE 2 ##STR4##

Compound 1 (100 mg; synthesis described in Example 1) is dissolved indry DMF (1.0 ml), 6 aminocaproic acid (29.3 mg; 1.0 eg) is then addedand the resulting mixture is stirred overnight at room temperature undernitrogen atmosphere. The following morning DCCI (50.7 mg; 1.1 eq) isadded, and the reaction mixture is stirred for an additional six hours.Solid precipitate (DCU) is removed by filtration, and the resulting DMFsolution is evaporated under reduced pressure to give a tacky solid,which is purified by flash chromatography upon silica gel (10%methanol/chloroform to give compound 2 (60 mg) as a white solid in 48%overall yield. (R=cyclohexylmethyl; n=2; X=6 aminocaproyl).

EXAMPLE 3 ##STR5##

Compound 2 (100 mg; synthesis described in Example 2) is dissolved indry DMF (2.0 ml), 6 aminocaproic acid (23.4 mg; 1.0 eg) is then added,and the resulting mixture is stirred overnight at room temperature undernitrogen atmosphere The following morning, DCCI (40.5 mg; 1.1 eq) isadded, and the reaction mixture is stirred for an additional six hours.Solid precipitate (DCU) is removed by filtration, and the resulting DMFsolution is evaporated under reduced pressure to give a tacky solid,which is purified by flash chromatography upon silica gel (10%methanol/chloroform) to give compound 3 (60.0 mg) as a white solid in50% overall yield. (R=cyclohexylmethyl; n=3; X=6-aminocaproyl).

EXAMPLE 4 ##STR6##

Compound 3 (100 mg; synthesis described in Example 3) is dissolved indry DMF (10.0 ml), 6-aminocaproic acid (19.5 mg; 1.0 eq) is then added,and the resulting mixture is stirred overnight at room temperature undernitrogen atmosphere. The following morining, DCCI (33.7 mg; 1.1 eq) isadded, and the reaction mixture is stirred for an additional six hours.Solid precipitate (DCU) is removed by filtration, and the resulting DMFsolution is evaporated under reduced pressure to give a tacky solid,which is purified by flash chromatography upon silica gel (10%methanol/chloroform) to give compound 4 (53 mg) as a white solid in 45%overall yield. (R=cyclohexylmethyl; n=4; X=6-aminocaproyl).

EXAMPLE 5 ##STR7##

CBZ-triglycine (4.0 g; Bachem Chem. Co.) is dissolved in 50.0 ml dryDMF. N-hydroxysuccinimide (1.42 g; 1.0 eg), and DCCI (2.55 g; 1.0 eg)are added and the resulting mixture is stirred overnight at roomtemperature under nitrogen atmosphere. The following precipitated DCU isremoved by filtration, and the resulting DMF solution is evaporatedunder reduced pressure to give a yellow oil. Recrystallization fromethyl acetate/chloroform gives the intermediate compound 7 (3.0 g) in57% yield. ##STR8##

Glycine t-butyl ester hydrochloride (0.54 g; Sigma Chem. Co.) issuspended in dry DMF (25.0 ml). Compound 7 (1.35 g; 1.0 eq) from Part a)is then added, along with triethylamine (1.62 g; 5.0 eg). The resultingsolution is allowed to stir overnight at room temperature under nitrogenatmosphere. The following morning, solvent is removed under reducedpressure to give a crude product. Recrystallization from ethylacetate/chloroform gives intermediate compound 8 (0.95 g) in 68% yield.##STR9##

Compound 8 (0.95 g) from Part b is dissolved in dry methanol (300 ml).Glacial acetic acid (0.45 ml) is then added and the solution is purgedwith nitrogen for 15 minutes. Palladium on carbon (1.5 g; palladiumcontent 10%) is then carefully added, with stirring. A stream ofhydrogen gas is bubbled through the stirring solution for three hours atroom temperature. The solution is carefully purged with nitrogen for 15minutes, then filtered. The filtrate solution is concentrated underreduced pressure to give intermediate compound 9 (700 mg) as the acetatesalt. ##STR10##

Compound 9 (700 mg: acetate salt) from Part c is dissolved in dry DMF(25 ml). N-(4-carboxycyclohexylmethyl)maleimide (697 mg) from Example 1is then added, and the mixture is allowed to stir overnight at roomtemperature under nitrogen atmosphere. The following morning DMF isevaporated under reduced pressure to afford a crude product.Recrystallization from ethyl acetate/hexane affords intermediatecompound 10 in 22% yield. ##STR11##

Compound 10 (225 mg) from Part d is suspended in chloroform (1.5 ml).Dry trifluoroacetic acid (1.5 ml) is then added, and the mixture isstirred at room temperature under a nitrogen atmosphere for a period ofthree hours. Solvent is evaporated under reduced atmosphere to give acrude product. Trituration with ethyl acetate gives intermediatecompound 11 (127 mg) in 61% yield. ##STR12##

Compound 11 (100 mg) from Part e is dissolved in dry DMF (7.0 ml) alongwith N-hydroxysuccinimide (37.1 mg; 1.5 eg) and DCCI (221.5 mg; 5.0 eq).The reaction mixture is stirred overnight at room temperature under anitrogen atmosphere. The following morning, precipitated DCU is removedby filtration, and DMF is evaporated under reduced pressure to give acrude solid. Trituration with chloroform gives compound 12 (86 mg) in60% yield. (R=cyclohexylmethyl; n=4; X=glycyl).

Compound 12 can be used for conjugating proteins (e.g. antibody andenzymes) to solid phases using the procedures outlined in the followingExamples.

EXAMPLE 6 ##STR13##

A round bottom flask equipped with a magnetic stirrer is charged withm-maleimidobenzoyl-N-hydroxysuccinimide ester (0.314 g; 0.001 mole)obtained from Pierce Corporation dissolved in DMF (5.0 mL).6-Aminocaproic acid (0.131 g; 1 equiv.) is added, and the resultingsolution is stirred overnight at room temperature under nitrogen. After18 hours, olicyclohexylcarbodiimide (DCCI; 0.206 g; 1.1 equiv.) is addedfollowed by N-hydroxysuccinimide (0.115 g, 1 equiv.). The reactionsolution is stirred for additional eight hours at room temperature undernitrogen. Precipitated dicyclohexylurea (DCU) is removed by filtration,and the resulting DMF solution is evaporated under reduced pressure. Theresulting solid is purified by silica gel chromatography (5% methanol inchloroform) to give compound 13 in 50% yield. This compound is treatedwith aminocaproic acid in a manner identical to the method described inexamples 2, 3 and 4 of this application to produce compounds where n isup to ten, and R=phenyl).

EXAMPLE 7 Preparation of Monoclonal anti-CA-125 IgG-DerivatizedMicroparticles using a 30 Atom Linkage a) Pretreatment of AmineMicroparticles

Amine microparticles (Seradyn; 0.164 micron; 2.5% solids; 1 ml) aremixed with 0.5 g Biorex ion exchange resin (Biorex MSZ501D; 20-50 mesh;catalog #142-7425). The mixture is rotated end-over-end for one hour atroom temperature, then vacuum filtered through a course sintered glassfunnel with washing. The filtrate is collected and centrifuged at 15,000rpm for 30 minutes. Supernatant is discarded, and the microparticlepellet is resuspended in distilled water with vortexing, and adjusted to2.5% solids by addition of distilled water.

b) Derivatization of the Particles

Resuspended, pre treated particles from part a at 2.5% solids are mixedwith an equal volume of compound 3 (Example 3) solution (2 mg/ml in DMF)and allowed to react at room temperature for one hour with end-over-endrotation. The reaction mixture is then diluted ten-fold withphosphate-buffered saline "PBS" (pH 7.2), and centrifuged at 15,000 rpmfor 30 minutes. The resulting supernatant is discarded, and themicroparticle pellet is resuspended with phosphate buffered saline. Thecentrifugation-resuspension sequence is repeated twice, the solution isagain centrifuged, supernatant is discarded, and the pellet isresuspended to a concentration of 2.5% solids with Tris buffer (0.05 Mtris; 0.1M NaCl; pH 8.0).

c) Preparation of the Antibody

A solution of monoclonal anti-CA-125 IgG (7.4 mg/ml; in phosphatebuffered saline) is incubated with DTT (dithiothreitol; 25 mM in finalreaction mixture) for twenty minutes at room temperature with stirringon a rotary agitator. The solution of partially reduced antibody is thendesalted by chromatography upon a pre-equilibrated Sephadex G-25(coarse) column with pH 7.0 phosphate buffer (0.1 M phosphate; 0.1MNaCl, 5 mM EDTA) as eluent. Fractions are collected, protein-containingfractions are pooled, and the protein concentration of the pooledsolution is estimated by measuring absorbance at 280 nm. cl d) Reactionof Maleimide-Derivatized Microparticles with Partially Reduced IgG

The partially reduced antibody from part c (1 ml; 1 mg/ml) is combinedwith the maleimide derivatized microparticles from part b) (1 ml; 2.5%solids). The mixture is rotated end over end at room temperatureovernight. The following morning, the reaction mixture is dilutedten-fold with wash buffer (0.01 M phosphate; pH 7.2; 1% Tween), thencentrifuged at 15,000 rpm for 30 minutes. The resulting supernatant isdiscarded, the microparticle pellet is washed twice (vortexing in 1 mlwash buffer; diluting tenfold with wash buffer, then centrifuging at15,000 rpm for 30 minutes followed by discarding supernatant). Thewashed pellet is resuspended to a final concentration of 0.125% solidsin storage buffer (0.01 M Tris; pH 8.1; 0.1M NaCl; 0.1% sodium azide;13.6% sucrose). The final microparticle suspension is first passedthrough a 23, then a 25 gauge needle. The resulting microparticleconjugate has the anti-CA-125 IgG antibody conjugated to themicroparticle with a 30 atom linker arm from Example 3. Microparticlesin storage buffer are stored until future use in an immunoassay for thedetection of CA- 125 antigen.

EXAMPLE 8 Preparation of Monoclonal anti-CA-125 IgG-DerivatizedMicroparticles using a 23 Atom Linkage

The procedure of Example 7 is repeated using the 23 atom linker group(Compound 2) from Example 2 instead of the 30 atom group of Example 7.

EXAMPLE 9

Preparation of Monoclonal anti-CA-125 IgG-Derivatized Microparticlesusing a 16 Atom Linkage

The procedure of Example 7 is repeated using the 16 atom linker group(Compound 1) from Example 1 instead of the 30 atom group of Example 7.

EXAMPLE 10 Preparation of Polyclonal anti-CA-125 IgG-DerivatizedMicroparticles using a 30 Atom Linkage

The procedure of Example 7 is repeated using a polyclonal anti-CA-125IgG antibody instead of the monoclonal antibody of Example 7. Polyclonalanti-CA-125 antibody was obtained by immunizing sheep subcutaneously andintramuscularly with 50,000 units of CA-125 antigen in Freund'sadjuvant. All subsequent boosts were done every two weeks using 50,000units of CA-125 antigen in Freund's incomplete adjuvant.

EXAMPLE 11

Preparation of Monoclonal anti-PAP IgG-Derivatized Microparticles usinga 30 Atom Linkage

a) Preparation of Reduced anti-PAP antibody

Dithiothreitol (DTT; 1.93 mg) is placed in a reaction vial. Mousemonoclonal anti-PAP antibody (0.5 ml; 6.98 mg/ml) is then added, and themixture is uncubated at room temperature for 20 minutes at roomtemperature. The reaction mixture is then applied to a pre-equilibratedSephadex G-25 column (coarse 1×20 cm) and eluted with pH 7.2 phosphatebuffer (0.2 M phosphate; 0.1M NaCl; 5.0 mM EDTA). Fractions arecollected and absorbance at 280 nm is measured. Protein-containingfractions are combined, the pool is diluted ten-fold with chromatographybuffer, and protein concentration of the resulting diluted pool ofactivated antibody is estimated by measuring absorbance at 280 nm.

b) Reaction of Derivatized Microparticles with Reduced Anti-PAP Antibody

Reduced antibody from part a (0 5 mg (e.g. 336 ul at 1.49 mg/ml)) isplaced in a reaction vial. The derivatized microparticle solution (0.5ml) from Example 7 part b is then added, and the mixture is incubatedovernight at room temperature on a rotary agitator. The followingmorning, the reaction mixture is centrifuged (20 minutes at 12,000 rpm),supernatant is removed, and absorbance at 280 nm is measured. Theantibody-derivatized microparticles are then resuspended in PBS-Tweenbuffer (0.1 g Tween in 100 ml PBS; 1.0 ml). The mixture is againcentrifuged (20 minutes at 12,000 rpm), supernatant is discarded, and5.0 ml storage buffer (0.01 M Tris; 0.1M NaCl; 0.1% sodium azide; 13.6%sucrose; pH 8.1) is then added. The microparticles are passed firstthrough a 23 gauge needle, then a 25 gauge needle. The resultingmicroparticle suspension is then transferred to a 10 ml plasticscrew-cap vial for storage until use in an enzyme immunoassay for thedetection of PAP antigen.

EXAMPLE 12

Covalent Attachment of B-12 Intrinsic Factor to Aminated PolystyreneMicroparticles

A 10% solution of 100 ul of aminated polystyrene microparticles (0.164micron, purchased from Seradyn) is placed in a reaction vial. A solutionof Compound 2 in DMF (9 ul; 1.21 mM) is added with a solution of B-12intrinsic factor (210 ul; 0.1537 mg/ml) in phosphate-buffered saline.The reaction mixture is rotated end-over-end overnight at roomtemperature. The following morning, the reaction mixture is centrifuged(30 minutes; 13,000 rpm). Supernatant is discarded, the remaining solidis resuspended in distilled water, and the centrifuging-resuspensionstep is repeated.

The product produced is a B-12 intrinsic factor/microparticle conjugatelinked with a 23 atom linker of Example 2. The particles are thensuspended in 750 ul of buffer (0.01 M Tris; 0.1M NaCl; 0.1% sodiumazide; 13.6% sucrose; pH 8.1) and used in an assay for the detection ofB-12.

EXAMPLE 13 Covalent Attachment of Recombinant Hepatitis B Core Antigento Aminated Polystyrene Microparticles Using Compound 3 a)Derivitization of Microparticles with Compound 3

Amino microparticles (Polysciences; 0.5 micron) are placed in a reactionvial. A solution of Compound 3 (Example 3) in DMF is then added, and themixture is treated as described in Example 7 part b.

b) Reaction of Recombinant Core Antigen With Derivatized Microparticles

Recombinant hepatitis B core antigen is placed in a vial. Derivatizedmicroparticles (from part a) are then added, and the mixture is treatedas decribed in Example 7 part d to yield microparticles conjugated tothe antigen with a 30 atom linker which can be used in assays fordetection of hepatitis B.

Example 14 Preparation of E. coli β-Galactosidase-DerivatizedMicroparticles With A 30 Atom Linkage

E. coli β-galactosidase (1 ml; 1 mg/ml) in pH 7.0 phosphate bufferedsaline (0.1M phosphate; 0.1M NaCl) is added to maleimide-derivatizedmicroparticles from Example 7, part b. The reaction mixture is rotatedend-over-end overnight at room temperature. The following morning thereaction mixture is centrifuged at 15,000 rpm for 30 minutes. Theresulting supernatant is discarded, and the microparticle pellet isresuspended to 2.5% solids with pH 7.0 phosphate-buffered saline (0.1Mphosphate; 0.1M NaCl). The centrifugation, decanting, resuspensionsequence is repeated twice. The microparticle pellet is finallyresuspended in storage buffer (0.1M tris; pH 7.0; 0.1M NaCl; 0.1% sodiumazide; 13.6% sucrose). The resulting microparticle suspension is passedthrough first a 23, then a 25 gauge needle. The product is E. coliβ-galactosidase conjugated to amino microparticles with a 30 atomlinkage. Enzyme derivatized microparticles in storage buffer are thenstored until future use.

EXAMPLE 15 Preparation of Calf Intestinal AlkalinePhosphatase-Derivatized Microparticles Using a 30 Atom Linkage a)Thiolation of the Enzyme

Calf intestinal alkaline phosphatase (0.5 ml; 10 mg/ml) in pH 8.0 Trisbuffer (0.05M Tris; 10 mM MgCl₂ ; 0.1 mM ZnCl₂) is placed in a reactionvial. Iminothiolane hydrochloride is then added to a concentration of4.0 mM. The mixture is stirred for 30 minutes at room temperature, thendesalted on a Sephadex G-25 (coarse) column with phosphate-bufferedsaline (0.1M phosphate; 0.1M NaCl; 10 mM MgCl₂, 0.1M ZnCl₂ ; pH 7.0) aseluent. Fractions are collected, protein-containing fractions arepooled, and protein concentration of the pooled solution of thiolatedenzyme is estimated by measuring absorbance at 280 nm.

b) Reaction of Thiolated Enzyme with Maleimide DerivatizedMicroparticles

Thiolated enzyme from part a (1 ml; 1 mg/ml) is combined with themaleimide-derivatized microparticles from Example 7, part b (1 ml; 2.5%solids). The reaction mixture is rotated end-over-end overnight at roomtemperature, then treated as described in Example 7, part d to provide asuspension of microparticles covalently derivatized with calf intestinalalkaline phosphatase.

EXAMPLE 16 Preparation of Monoclonal Anti-CA-125 IgG-DerivatizedMicroparticles a) Preparation of Thiolated Microparticles

Resuspended, pretreated amine microparticles from Example 7, part a (1ml; 2.5% solids) are mixed with iminothiolane HCl to achieve a finaliminothiolane concentration of 50 mM. The reaction mixture is stirred atroom temperature for one hour, then treated as described in Example 7,part b.

b) Derivatization of the Antibody

A solution of monoclonal anti-CA-125 IgG (7.4 mg/ml) inphosphate-buffered saline is incubated with 30 molar equivalents of aDMF solution of Compound 3 (5.0 mM). The reaction mixture is stirred for30 minutes at room temperature, then desalted on a Sephadex G-25(coarse) column with pH 7.0 phosphate buffer (0.1M phosphate; 0.1M NaCl)as eluent. Fractions are collected, protein containing fractions arepooled, and protein concentration of the pooled solution is estimated bymeasuring absorbance at 280 nm.

c) Reaction of Thiolated Microparticles with Maleimide-DerivatizedAntibodies

Thiolated microparticles from part a (1 ml; 2.5% solid) are mixed withmaleimide derivatized antibodies from part b) (1 ml; 1 mg/ml). Thereaction mixture is rotated end-over-end overnight at room temperature.The following morning, the antibody-derivatized microparticles aretreated as described in Example 7, part d) to produce amicroparticle/IgG conjugate which can be used in an assay for thedetection of CA-125 antigen.

EXAMPLE 17 Preparation of Monoclonal anti-CA-125 IgG-Derivatized NylonFibers a) Pretreatment of Nylon Fibers

Nylon monofilement fishing line (Berkley, 6 inches, 2 lb. test) isincubated for 30 minutes at room temperature with 3N HCl (10 ml) withshaking. The fiber is then washed twice with 20 ml distilled water. Thewashed, partially hydrolyzed fiber is stored in distilled water untilfurther use.

b) Maleimide Derivitization of Partially Hydrolyzed Nylon Fiber

A two inch section of partially hydrolyzed nylon fiber from part a) iscut into 1/8 inch pieces. The 1/8 inch nylon lengths are placed in areaction vial along with a DMF solution of Compound 3 (1.0 ml; 5.0 mM).The reaction mixture is shaken vigorously for two hours, then filteredthrough a course sintered glass funnel. The maleimide derivatized nylonfibers are washed several times with distilled water, then stored untilfurther use in distilled water.

c) Reaction of Maleimide Derivatized Nylon Fibers with Partially Reducedanti-CA-125 IgG

Partially reduced monoclonal anti-CA-125 IgG from Example 7, part c) (1ml; 1 mg/ml) is added to maleimide-derivatized nylon fibers from part b.The reaction mixture is incubated overnight at room temperature withend-over-end rotation. The following morning, the reaction mixture isfiltered through a coarse sintered glass funnel and theantibody-derivatized fibers are washed several times with wash buffer(0.1M phosphate; 0.1M NaCl; pH 7.0). The resulting fibers containcovalently attached monoclonal anti-CA-125 IgG via a 30 atom spacergroup. The resulting fibers are stored in fiber storage buffer (0.1M,phosphate; 0.1M NaCl; 1% BSA; 0.1% sodium azide) for use in an assay forthe detection of CA-125 antigen).

EXAMPLE 18 Preparation of Monoclonal anti-CA-125 IgG-Derivatized WoolFibers a) Partial Reduction of Wool Thread

Wool thread is cut into 1/2 inch pieces. A piece of thread is thenimmersed in 1 ml 5 mM DTT solution and shaken vigorously for one hour.The reaction mixture is then filtered through a coarse sintered glassfunnel, and washed five times with buffer (pH 7.0; 0.1M phosphate; 0.1MNaCl; 5 mM EDTA).

b) Reaction of Maleimide-Derivatized Antibody With Partially ReducedWool Thread

Partially reduced wool thread from part a is placed in a reaction vial.Maleimide-derivatized monoclonal anti-CA-125 IgG from Example 7, part d(1 ml; 1 mg/ml) is then added, and the mixture is rotated end-over-endovernight at room temperature. The following morning, the reactionmixture is filtered through a coarse sintered glass funnel and washedfive times with wash buffer (0.1M phosphate; 0.1M NaCl; pH 7.0). Theresulting fiber contains monoclonal anti-CA-125 IgG covalently attachedvia a 30 atom spacer group. The resulting fibers are stored in fiberstorage buffer (0.1M phosphate; 0.1M NaCl; 1% bovine serum albumin; 0.1%sodium azide) until used in an assay for the detection of CA-125antigen.

What is claimed is:
 1. A chemically derivatized solid phases materialcomprising ##STR14## wherein B is an amine bearing solid phase material;X is an amino acid having from three to ten carbon atoms in a straightchain; n is from one to ten; and R is an alkyl, cycloalkyl, analkyl-cycloalkyl or an aromatic carbocyclic ring.
 2. The chemicallyderivatized solid phase material of claim 1 wherein R iscyclohexylmethyl.
 3. The chemically derivatized solid phase material ofclaim 1 wherein B is an amine bearing microparticle.
 4. The chemicallyderivatized solid phase material of claim 1 wherein said solid phasecomprises a fiber.
 5. The chemically derivatized solid phase material ofclaim 1 wherein X includes aminocaproyl.